Renal epithelial cells, particularly cells of the inner medulla, are often exposed to adverse stress including hyperosmolality, low oxygen tension and nephrotoxins. Survival of such adverse stimuli requires over-expression of evolutionarily conserved stress genes (e.g., heat shock proteins) that function as molecular chaperones to assist in protein synthesis and folding. Recently our laboratory cloned and sequenced a new cDNA, known as Osmotic Stress Protein, 94 kDa (Osp94) which is a member of the superfamily of molecular chaperone/heat shock proteins. Osp94 shows greatest homology to Hsp110 and Hsp70RY, members of the newly discovered Hsp110 subfamily. The proteins encoded by Osp94, Hsp110, and Hsp70RY possess an N-terminal ATP-binding domain and a C-terminal peptide-binding domain characteristic of molecular chaperones. These genes are ubiquitously expressed and can be greatly up-regulated by hyperosmolar NaCl and other stresses. The overall objective of this project is to characterize the regulation and biological function of these three genes. Using mIMCD3 cells we will address Jour Specific Aims: l) Define at the mRNA level the responses of Osp94, Hsp110, and Hsp70RY to stresses such as hyperosmolar NaCl, heat shock, heavy metal toxicity, and ATP depletion. 2) Define at the protein level the responses of these three genes to stress by creating specific antibodies for western and immunohistochemical analyses. 3) For Osp94 only, establish that stress-induced gene expression involves increased transcription and define the gene promoter element(s) that are responsible. Following nuclear run-on analysis we will perform luciferase reporter assays using deletion/truncation constructs from the 5' flanking region of Osp94 which we have already isolated. 4) Characterize the chaperone properties of Osp94, Hsp110, and Hsp70RY by using an unfolded protein affinity column that binds molecular chaperones in an ATP-dependent manner. By comparing and contrasting the regulation and chaperone function of these genes we will gain greater insight into their roles in physiological and pathophysiological states.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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General Medicine B Study Section (GMB)
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Brigham and Women's Hospital
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Kojima, Ryoji; Randall, Jeffrey D; Ito, Eri et al. (2004) Regulation of expression of the stress response gene, Osp94: identification of the tonicity response element and intracellular signalling pathways. Biochem J 380:783-94
Santos, Bento C; Pullman, James M; Chevaile, Alejandro et al. (2003) Chronic hyperosmolarity mediates constitutive expression of molecular chaperones and resistance to injury. Am J Physiol Renal Physiol 284:F564-74
Borkan, Steven C; Gullans, Steven R (2002) Molecular chaperones in the kidney. Annu Rev Physiol 64:503-27
Yoshida, Takumi; Kurella, Manjula; Beato, Francisca et al. (2002) Monitoring changes in gene expression in renal ischemia-reperfusion in the rat. Kidney Int 61:1646-54
Santos, B C; Chevaile, A; Hebert, M J et al. (1998) A combination of NaCl and urea enhances survival of IMCD cells to hyperosmolality. Am J Physiol 274:F1167-73
Santos, B C; Chevaile, A; Kojima, R et al. (1998) Characterization of the Hsp110/SSE gene family response to hyperosmolality and other stresses. Am J Physiol 274:F1054-61